High throughput screening has become a dominant tool in the pharmaceutical industry for the discovery of lead compounds that can be modified into candidates for drug development. For instance, it is abundantly used for identification of ligands with high affinity for receptors. In this regard, combinatorial techniques have provided approaches to generating and deconvoluting large libraries of test compounds in high throughput screens. It involves selection and amplification of a subset of molecules with desired biological properties from complex libraries.
One technique which has emerged for identification of peptide leads involves the use of peptide display methodologies such as phage display. Phage-displayed peptide libraries can comprise vast collections of short, randomized polypeptides that are displayed on the surface of a filamentous bacteriophage particle. Thus, each xe2x80x9cpeptidexe2x80x9d is actually the N-terminal sequence of a phage-coat protein, that is encoded by a randomly-mutated region of the phage genome responsible for the production of the coat protein. In this manner, each unique peptide in the library is physically linked with the DNA molecule encoding it. Antibodies and other binding molecules can be used as xe2x80x9ctargetsxe2x80x9d to specifically select rare phage clones bearing ligand peptides, and sequencing of the corresponding viral DNA will reveal their amino acid sequences. Relatively high-affinity peptides for a variety of peptide- and non-peptide-binding targets have been affinity-isolated from epitope libraries. This technology has been used to map epitopes on proteins and to find peptide mimics for a variety of target molecules. Many powerful applications can be envisioned in the areas of drug design and the development of diagnostic markers, vaccines and toleragens.
For the purposes of drug discovery, there are potential advantages in the use of genetically encoded libraries, such as phage display (Scott et al, Science 249, 386 (1990); Devlin et al., Science 249, 386 (1990)), xe2x80x9cpeptide on plasmidxe2x80x9d (Cull et al. PNAS 89, 1865 (1992)), and in vitro translation-based systems (Mattheakis et al. PNAS 91, 9022 (1994)), compared to the use of synthetic small molecule libraries (Bunin et al. PNAS 91, 4708 (1994); Gordon et al. J. Med. Chem. 37, 1385 (1994); and Dooley et al., Science 266, 2019 (1994)). The genetic encoding of libraries allows the resynthesis and rescreening of molecules with a desired binding activity. The resulting amplification of interacting molecules in subsequent rounds of selection can lead to the isolation of extremely rare, specific binders from a large pool of molecules.
However, despite the success of these methods, they suffer from numerous sources of error and bias, such as very low initial concentrations of species, non-specific binding, and, significantly, the sampling of only a fraction of the library at the end of an experiment.
One aspect of the invention provides a method for generating a peptide with a selected biological activity, comprising the steps of:
(i) providing a peptide display library comprising a variegated population of test peptides expressed on the surface of a population of display packages;
(ii) in a display mode, isolating, from the peptide display libary, a sub-population of display packages enriched for test peptides which have a desired binding specificity and/or affinity for a cell or a component thereof;
(iii) in a secretion mode, simultaneously expressing the enriched test peptide sub-population under conditions wherein the test peptides are secreted and are free of the display packages; and
(iv) assessing the ability of the secreted test peptides to regulate a biological process in a target cell.
For instance, the peptide display library can be a phage display library, e.g., which utilizes phage particles such as M13, f1, fd, If1, Ike, Xf, Pf1, Pf3, xcex, T4, T7, P2, P4, xcfx86X-174, MS2 or f2. In preferred embodiments, the phage display library is generated with a filamentous bacteriophage specific for Escherichia coli and the phage coat protein is coat protein III or coat protein VIII. For instance, the filamentous bacteriophage can be M13, fd, and f1.
In other embodiments, the peptide display library is a bacterial cell-surface display library or a spore display library.
In certain embodiments, the test peptides are enriched from the peptide display library in the display mode by a differential binding means comprising affinity separation of test peptides which specifically bind the cell or component thereof from test peptides which do not. For example, the differential binding means can include panning the peptide display library on whole cells, affinity chromatographic means in which a component of a cell is provided as part of an insoluble matrix (.e.g, a cell surface protein attached to a polymeric support), and/or immunoprecipitating the display packages.
In the display mode, the test peptides can be enriched for those which bind to a cell-type specific marker and/or a cell surface receptor protein. For example, the test peptide library can be enriched in the display mode for test peptides which bind to a G-protein coupled receptor, such as a chemoattractant peptide receptor, a neuropeptide receptor, a light receptor, a neurotransmitter receptor, a cyclic AMP receptor, or a polypeptide hormone receptor. In other embodiments, the test peptide library can be enriched in the display mode for test peptides which bind to a receptor tyrosine kinase, such as an EPH receptor. In still other embodiments, the test peptide library can be enriched in the display mode for test peptides which bind to a cytokine receptor or an MIRR receptor. In certain embodiments, the test peptide library can be enriched in the display mode for test peptides which bind to an orphan receptor.
In preferred embodiments, the peptide display library includes at least 103 different test peptides.
In preferred embodiments, the test peptides are 4-20 amino acid residues in length.
In certain embodiments, each of the test peptides are encoded by a chimeric gene comprising (i) a coding sequence for the test peptide, (ii) a coding sequence for a surface protein of the display package for displaying the test peptides on the surface of a population of display packages, and (iii) RNA splice sites flanking the coding sequence for the surface protein, wherein, in the display mode, the chimeric gene is expressed as fusion protein including the test peptide and the surface protein, whereas in the secretion mode, the test peptide is expressed without the surface protein as a result of the coding sequence for the surface protein being removed by RNA splicing.
In preferred embodiments, the test peptides are expressed by a eukaryotic cell, more preferably a mammalian cell, in the secretion mode.
In preferred embodiments, the target cell is a eukaryotic cell, more preferably a mammalian cell such as a human cell.
In certain embodiments, the biological process scored for in the secretion mode includes a change in cell proliferation, cell differentiation or cell death. In other embodiments, the biological process which is detected is changes in intracellular calcium mobilization, intracellular protein phosphorylation, phospholipid metabolism, and/or expression of cell-specific marker genes.
In certain embodiments, the target cell includes a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the signal transduction acitivity of the cell surface receptor protein, expression of the reporter gene providing the detectable signal. For instance, the reporter gene can encode a gene product that gives rise to a detectable signal selected from the group consisting of: color, fluorescence, luminescence, cell viability relief of a cell nutritional requirement, cell growth, and drug resistance. In preferred embodiments, the reporter gene encodes a gene product selected from the group consisting of chloramphenicol acetyl transferase, beta-galactosidase and secreted alkaline phosphatase. In other preferred embodiments, the reporter gene encodes a gene product which confers a growth signal.
In certain embodiments, the secretion mode includes assessing the ability of the secreted test peptides to inhibit the biological activity of an exogenously added compound on the target cells.
In an exemplary embodiment: in step (ii) above, display packages which bind to endothelial cells are isolated; and in step (iv) above, the ability of the secreted test peptides to inhibit proliferation of endothelial cells is assessed. For example, in step (iv) the ability of the secreted test peptides to inhibit proliferation of endothelial cells in the presense of an angiogenic amount of an endogenous growth factor can be assessed.
The subject invention also specifically contemplates that peptides identified in the secretion mode can be converted into peptidomimietics.
Moreover, in certain embodiments, the subject method includes the further step of formulating, with a pharmaceutically acceptable carrier, one or more test peptides which regulate the biological process in the target cell or peptidomimetics thereof.
Another aspect of the present invention provides a peptide display library enriched for test peptides having a desired binding specificity and/or affinity for a cell or a component thereof and which regulate a biological process in a target cell.
Still another aspect of the present invention relates to a vector comprising a chimeric gene for a chimeric protein, which chimeric gene comprises (i) a coding sequence for a test peptide, (ii) a coding sequence for a surface protein of a display package, and (iii) RNA splice sites flanking the coding sequence for the surface protein, wherein,
in a display mode, the chimeric gene is expressed as a fusion protein including the test peptide and the surface protein such that the test peptide can be displayed on the surface of a population of display packages,
whereas in the secretion mode, the test peptide is expressed without the surface protein as a result of the coding sequence for the surface protein being removed by RNA splicing.
In certain embodiments, the chimeric gene can include a secretion signal sequence for secretion of the test peptide in the secretion mode, e.g., secretion of the test peptide from eukaryotic cells, preferably mammalian cells.
Yet another aspect of the present invention provides a vector library, each vector comprising a chimeric gene for a chimeric protein, which chimeric gene comprises (i) a coding sequence for a test peptide, (ii) a coding sequence for a surface protein of a display package, and (iii) RNA splice sites flanking the coding sequence for the surface protein, wherein,
in a display mode, the chimeric gene is expressed as fusion protein including the test peptide and the surface protein such that the test peptide can be displayed on the surface of a population of display packages,
whereas in the secretion mode, the test peptide is expressed without the surface protein as a result of the coding sequence for the surface protein being removed by RNA splicing,
the vector library collectively encodes a variegated population of test peptides.
In preferred embodiments, the vector library collectively encodes at least 103 different test peptides.
In preferred embodiments, the test peptides are 4-20 amino acid residues in length.
Another aspect of the present invention is a cell composition comprising a population of cells containing the vector library described above.
Still another aspect of the present invention provides a method for generating a peptide with a selected antimicrobial activity, comprising the steps of:
(i) providing a recombinant host cell population which expresses a soluble peptide library comprising a variegated population of test peptides;
(ii) culturing the host cells with a target microorganism under conditions wherein the peptide library is secreted and diffuses to the target microorganism; and
(iii) selected host cells expressing test peptides that inhibit growth of the target microorganism.
For example, the target microorganism is a bacteria or a fungus. In certain embodiments, the host cells are cultured on agar embedded with the target microorganisms. For example, antimicrobial activity of a test peptide can be determined by zone clearing in the agar.